Role of uric acid in different types of calcium oxalate renal calculi

Authors

  • FÉLIX GRASES,

    1. Laboratory of Renal Lithiasis Research, Institute of Health Sciences Research (IUNICS), University of Balearic Islands, Mallorca, Spain
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  • PILAR SANCHIS,

    1. Laboratory of Renal Lithiasis Research, Institute of Health Sciences Research (IUNICS), University of Balearic Islands, Mallorca, Spain
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  • JOAN PERELLÓ,

    1. Laboratory of Renal Lithiasis Research, Institute of Health Sciences Research (IUNICS), University of Balearic Islands, Mallorca, Spain
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  • ANTONIA COSTA-BAUZÁ

    1. Laboratory of Renal Lithiasis Research, Institute of Health Sciences Research (IUNICS), University of Balearic Islands, Mallorca, Spain
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Félix Grases phd, Laboratory of Renal Lithiasis Research, Faculty of Sciences, University of Balearic Islands, 07122, Palma of Mallorca, Spain. Email: fgrases@uib.es

Abstract

Aim:  The presence of uric acid in the beginning zone of different types of ‘pure’ calcium oxalate renal calculi was evaluated with the aim of establishing the degree of participation of uric acid crystals in the formation of such calculi.

Methods:  The core or fragment of different types of ‘pure’ calcium oxalate renal calculi was detached, pulverized and uric acid extracted. Uric acid was determined using a high-performance liquid chromatography/mass spectrometry method.

Results:  In calcium oxalate monohydrate (COM) papillary calculi with a core constituted by COM crystals and organic matter, 0.030 ± 0.007% uric acid was found in the core. In COM papillary calculi with a core constituted by hydroxyapatite, 0.031 ± 0.008% uric acid was found in the core. In COM unattached calculi (formed in renal cavities) with the core mainly formed by COM crystals and organic matter, 0.24 ± 0.09% uric acid was found in the core. In COM unattached calculi with the core formed by uric acid identifiable by scanning electron microscopy (SEM) coupled to X-ray microanalysis, 20.8 ± 7.8% uric acid was found in the core. In calcium oxalate dihydrate (COD) unattached calculi containing little amounts of organic matter, 0.012 ± 0.004% uric acid was found. In COD unattached calculi containing little amounts of organic matter and hydroxyapatite, 0.0030 ± 0.0004% of uric acid was found.

Conclusions:  From these results it can be deduced that uric acid can play an important role as inducer (heterogeneous nucleant) of COM unattached calculi with the core formed by uric acid identifiable by SEM coupled to X-ray microanalysis (these calculi constitute the 1.2% of all calculi) and in COM unattached calculi with the core mainly formed by COM crystals and organic matter (these calculi constitute the 10.8% of all calculi).

Introduction

An important number of clinical and epidemiological studies relate hyperuricosuria to calcium oxalate stone formation.1–3 In fact, allopurinol has clearly demonstrated positive effects on decreasing the recurrence of calcium oxalate renal lithiasis.4–6

Uric acid crystals have crystallographic features similar to calcium oxalate monohydrate (COM) crystals, and for this reason they act on them as active inducers of COM heterogeneous nucleation.7,8 Consequently, this fact would explain the physicochemical mechanism by which uric acid affects calcium oxalate crystallization.

In spite of these facts, calcium oxalate/uric acid mixed calculi are not very common, corresponding to only 2.6% of all renal calculi.9 The aim of this paper is to study the presence of uric acid in the beginning zone of different types of ‘pure’ calcium oxalate renal calculi, to evaluate the degree of participation of uric acid crystals in the formation of such calculi.

Materials and methods

Urinary calculi

Fifty-nine ‘pure’ calcium oxalate renal calculi (calcium oxalate was the main component) were selected, belonging to six groups of our renal calculi classification:9

  • • Group 1: COM papillary calculi with the core constituted by COM crystals and organic matter (n = 9; Fig. 1a)
  • • Group 2: COM papillary calculi with the core constituted by hydroxyapatite (n = 11; Fig. 1b)
  • • Group 3: COM unattached calculi (formed in renal cavities) with the core formed by COM crystals and organic matter (n = 9; Fig. 1c)
  • • Group 4: COM unattached calculi with the core formed by uric acid identifiable by scanning electron microscopy (SEM) coupled to X-ray microanalysis (n = 10; Fig. 1d)
  • • Group 5: Calcium oxalate dihydrate (COD) unattached calculi containing little amounts of organic matter (n = 10; Fig. 1e)
  • • Group 6: COD unattached calculi containing little amounts of organic matter and hydroxyapatite (n = 10; Fig. 1f)
Figure 1.

Images by scanning electron microscopy of the different types of calculi used: (a) Calcium oxalate monohydrate (COM) papillary calculi with the core constituted by COM crystals and organic matter. (b) COM papillary calculi with the core constituted by hydroxyapatite. (c) COM unattached calculi (formed in renal cavities) with the core formed by COM crystals and organic matter. (d) COM unattached calculi with the core formed by uric acid identifiable by scanning electron microscopy coupled to X-ray microanalysis. (e) Calcium oxalate dihydrate (COD) unattached calculi containing little amounts of organic matter. (f) COD unattached calculi containing little amounts of organic matter and hydroxyapatite.

Stones classified as ‘pure’ calcium oxalate were selected among the non-fragmented calculi from our stone collection containing over 5000 specimens. The procedure used to analyse and study renal calculi requires an appropriate combination of observation by means of macroscopic and microscopic conventional techniques (stereoscopic microscope; Optomic, Madrid, Spain) with physical techniques as infrarred spectrometry (infrared spectroscope Bruker IFS 66; Bruker, Ettlingen, Germany) and scanning electron microscopy (Hitachi S-530; Hitachi, Tokyo, Japan) coupled to X-ray microanalysis (Oxford Link Isis; Oxford, UK).10

The study of COM calculi (papillary or unattached) begins through the direct observation of its external aspect, using a stereoscopic microscope. Afterwards, each calculus is sectioned into two parts along a plane as near as possible to its geometric center, in order to be able to establish the internal structure and to identify the core of the calculus. Afterwards, using a slender metallic needle, the core is separated from the rest of the calculus and the amount of uric acid in it was evaluated as it is explained below.

Calcium oxalate dihydrate calculi were characterized by the absence of established core and they were mainly constituted by interconnected bipyramidal COD crystals and little amounts of hydroxyapatite and/or organic matter between them (they can also contain variable amounts of COM, but it comes from the transformation of COD). Due to these types of structure, a representative fragment of each calculus was selected afterwards to evaluate the amount of uric acid in them, using the methodology explained below.

Uric acid analysis

Apparatus

Due to the necessity of a highly sensitive and selective method of uric acid analysis, a high-performance liquid chromatography/mass spectrometry (LC/MSD) method was selected.11 This analysis was performed with an Agilent 1100 Series LC/MSD system (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separations were performed at 25°C on a Zorbax Sax Column (150 mm × 4.6 mm i.d.), an anion exchange resin, 5 µm particle size (Agilent Technologies) with a 12.5 cm × 4.6 mm i.d. guard column (Agilent Technologies). The mobile phase (50% sodium citrate 1 mmol/L at pH = 6.5, 50% acetonitrile) was delivered at a flow rate of 1 mL/min. Mass spectral identification of uric acid was carried out with an electrospray ionization interface and a quadrupole mass analyser. The mobile phase was nebulized by nitrogen gas at 350°C, with a flow rate of 13 L/min, into an electrospray mass analyser. The detector counted negative ions with selected ion monitor (SIM) mode, by monitoring m/z = 167.1, which corresponds to the urate anion, the most abundant ion. The nebulization pressure used was 60 psi and the fragmentor voltage 80 V. Capillary voltage was 3000 V.

Treatment for renal calculi

The core or fragment of each calculus was detached, pulverized and uric acid extracted with 1 mL of NaOH at pH = 11. The extracts were filtered through 0.45 µm pore filters.

Samples of group 4 were 50-fold diluted due to the high amount of uric acid found in them.

In all cases, 10 µL of the solution was injected in the LC/MSD system.

Standards were prepared from aqueous solutions of uric acid and the analytical determination was carried out using the corresponding calibration curve.

Analysis of urinary samples

The urines of the stone-formers that generated the selected calculi were studied according to the following protocol. All subjects were on free diet at the time of urine collection and none of the stone-formers were undergoing pharmacological treatment of any kind. Twenty-four hour urine samples were collected in sterile flasks containing thymol as a preservative, and refrigerated immediately. After collection, the volume was recorded, the pH was measured immediately with a Crison pH meter (Crison, Barcelona, Spain), and the samples stored at −20°C until they were assayed. Normally, urine was collected 1–2 months after stone passage. Calcium, magnesium and phosphorus were determined by inductively coupled plasma atomic spectroscopy. Uric acid and creatinine were determined by means of a Roche Modular Analytics kit (Roche, Basel, Switzerland) with 11875426216 and 11875663216 reagents, respectively. Citrate and oxalate were determined by means of the R-Biopharm enzymatic test kits N°10139076035 and 10755699035, respectively (R-Biopharm, Darmstadt, Germany).

Statistics

Values in the table and figures are expressed as mean ± SE. One-way anova was used to calculate significance of difference between groups. The Student t-test was used to assess differences between means. Conventional Windows software was used for statistical computations. A value of P < 0.05 was considered to assess statistical significance.

Results

The uric acid amounts found in the six categories of ‘pure’ calcium oxalate renal calculi are shown in Table 1. As can be observed, as expected, the maximum amount was found in COM unattached calculi with the core formed by uric acid identifiable by SEM coupled to X-ray microanalysis (20.8 ± 7.8% of uric acid in core). When the rest of calculi were compared it can be seen that only the COM unattached calculi with the core mainly formed by COM crystals and organic matter contained significantly higher amounts of uric acid (0.24 ± 0.09%). In fact, the rest of calcium oxalate renal calculi showed similar amounts of uric acid, around 10 times inferior to the former (0.003–0.031%).

Table 1.  Percentage of uric acid (expressed as mean ± SE) found in the core (groups 1–4) or a fragment (groups 5, 6) of different types of selected ‘pure’ calcium oxalate renal calculi
GroupType of calculi% of uric acid (weight of uric acid [g]/weight of core or fragment [g]) × 100
  • a

    P = 0.0333 vs group 1;

  • b

    P = 0.0191 vs group 2;

  • c

    P = 0.0216 vs group 1;

  • d

    P = 0.0111 vs group 2;

  • e

    P = 0.0228 vs group 3;

  • f

    P = 0.0339 vs group 1;

  • g

    P = 0.0470 vs group 2;

  • h

    P = 0.0160 vs group 3;

  • i

    P = 0.0154 vs group 4;

  • j

    P = 0.0007 vs group 1;

  • k

    P = 0.0023 vs group 2;

  • l

    P = 0.0126 vs group 3;

  • m

    P = 0.0154 vs group 4;

  • n

    P = 0.0273 vs group 5. COD, calcium oxalate dehydrate; COM, calcium oxalate monohydrate; SE, standard error; SEM, scanning electron microscopy.

1COM papillary calculi with the core constituted by COM crystals and organic matter (n = 9; Fig. 1a) 0.030 ± 0.007
2COM papillary calculi with the core constituted by hydroxyapatite (n = 11; Fig. 1b) 0.031 ± 0.008
3COM unattached calculi (formed in renal cavities) with the core formed by COM crystals and organic matter (n = 9; Fig. 1c) 0.24 ± 0.09a,b
4COM unattached calculi with the core formed by uric acid identifiable by SEM coupled to X-ray microanalysis (n = 10; Fig. 1d) 20.8 ± 7.8c,d,e
5COD unattached calculi containing little amounts of organic matter (n = 10; Fig. 1e) 0.012 ± 0.004f,g,h,i
6COD unattached calculi containing little amounts of organic matter and hydroxyapatite (n = 10; Fig. 1f) 0.0030 ± 0.0004j,k,l,m,n

The common urinary biochemical data (mean ± SE) of all six groups are shown in Table 2. As can be expected, hypercalciuria was observed only in the group of stone formers that generated COD calculi. It is interesting to observe that urinary pH of the group of stone-formers that generated COM unattached calculi with the core formed by COM crystals and organic matter was 5.5 ± 0.1 and the urinary pH of the group of stone-formers that generated COM unattached calculi with the core formed by uric acid identifiable by SEM coupled to X-ray microanalysis was 5.6 ± 0.2.

Table 2.  Urinary biochemical parameters (expressed as mean ± SE) for the stone-formers that generated the different types of selected ‘pure’ calcium oxalate renal calculi
 Groups
123456
  1. Group 1: stone-formers that generated COM papillary calculi with the core constituted by COM crystals and organic matter (n = 9); Group 2: stone-formers that generated COM papillary calculi with the core constituted by hydroxyapatite (n = 11); Group 3: stone-formers that generated COM unattached calculi (formed in renal cavities) with the core formed by COM crystals and organic matter (n = 9); Group 4: stone-formers that generated COM unattached calculi with the core formed by uric acid identifiable by SEM coupled to X-ray microanalysis (n = 10); Group 5: stone-formers that generated COD unattached calculi containing little amounts of organic matter (n = 10); Group 6: stone-formers that generated COD unattached calculi containing little amounts of organic matter and hydroxyapatite (n = 10). COD, calcium oxalate dihydrate; COM, calcium oxalate monohydrate; SE, standard error; SEM, scanning electron microscopy.

Diuresis (mL) 1800 ± 180 1670 ± 112 1853 ± 140 1679 ± 117 1430 ± 90 1630 ± 116
pH 5.7 ± 0.1 6.2 ± 0.3 5.5 ± 0.1 5.6 ± 0.2 5.6 ± 0.1 5.7 ± 0.2
Creatinine (mg/L) 1011 ± 97 849 ± 73 843 ± 64 890 ± 94 1019 ± 135 1033 ± 90
Calcium (mg/L) 108 ± 11 155 ± 12 86 ± 8 106 ± 8 197 ± 18 234 ± 15
Magnesium (mg/L) 59 ± 5 56 ± 4 51 ± 5 55 ± 4 66 ± 9 62 ± 4
Phosphorous (mg/L) 544 ± 57 526 ± 75 481 ± 48 553 ± 65 620 ± 69 672 ± 68
Oxalate (mg/L) 18 ± 2 16 ± 2 18 ± 1 19 ± 2 21 ± 8 18 ± 2
Uric acid (mg/L) 430 ± 42 386 ± 38 381 ± 40 420 ± 34 485 ± 62 421 ± 43
Citrate (mg/L) 377 ± 41 491 ± 31 418 ± 39 379 ± 56 405 ± 67 432 ± 47

Discussion

Undoubtedly, uric acid has an important contribution to the formation of calcium oxalate/uric acid mixed calculi. Nevertheless, this group of renal calculi constitutes only 2.6% of all renal calculi,9 and this is in clear disagreement with epidemiologic studies that relate a high incidence of hyperuricosuria to calcium oxalate stone formation.1–3 Nevertheless, from the results presented in this paper, it can be deduced that in a significant number of COM unattached calculi, uric acid can also play an important role as an inducer (heterogeneous nucleant) of COM crystal development. Thus, the contribution of uric acid crystals to the calculi formation for COM unattached calculi with the core formed by uric acid identifiable by SEM coupled to X-ray microanalysis is obvious. However, it must be emphasized that when applying conventional infrared spectrometry analysis to these calculi, uric acid is not detected in the majority of occasions. On the other hand, it is clear that in COM unattached calculi with the core mainly formed by COM crystals and organic matter, the significantly high amounts of uric acid found in the core in comparison to the other types of ‘pure’ calcium oxalate renal calculi also allows us to attribute to these crystals an important role in the generation of this type of calculi. Thus, considering that in some of these calculi uric acid is clearly not implied in their formation, like COD unattached calculi containing little amounts of organic matter and hydroxyapatite (in which hydroxyapatite acts as a heterogeneous nucleant12), it can be assumed that in the other types in which it is present at similar amounts, uric acid also does not exert any important action. Hence, the formation of little amounts of uric acid crystals would induce the heterogeneous nucleation of COM crystals on them when they are retained in renal cavities with urodynamic deficiency (intrapelvic spaces or renal pelvis). In this case, the formation of uric acid crystals is justified considering the urinary pH found in these patients (Table 2), since uric acid solubility decreases abruptly at urinary pH < 5.5. These uric acid crystals (acting as heterogeneous nucleants of COM crystals) represent the first and main stage of these uric acid-induced COM unattached calculi, and the regular growth of new COM crystals on the surface of the already formed objects generate the core of the calculus. The formation of the core is the first and perhaps the most important step in COM unattached calculus development. This core serves later as a substrate for the growth of columnar COM crystals constituting the compact striated layer of the COM stone. Taking into account that COM unattached calculi with the core mainly formed by COM crystals and organic matter constitute the 10.8% of all calculi, the COM unattached calculi with the core formed by uric acid identifiable by SEM coupled to X-ray microanalysis constitute the 1.2% of all calculi9 and if 2.6% of calcium oxalate/uric acid mixed calculi is also considered, it can be deduced that uric acid would be implied in approximately 15% of all calculi.

Acknowledgments

J.P. and P.S. express their appreciation to the Spanish Ministry of Education, Culture and Sport for a fellowship of the FPU program. The financial support from Conselleria d’Innovació i Energia, Govern Balear (Grant PROIB-2002GC1-04) and (project BQU 2003-01659) from the Spanish Ministry of Science and Technology is gratefully acknowledged.

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